Turnover is rate-limited by deglycosylation for Micromonospora viridifaciens sialidase-catalyzed hydrolyses: conformational implications for the Michaelis complex

A panel of seven isotopically substituted sialoside natural substrate analogues based on the core structure 7-(5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonic acid)-(2→6)-β-D-galactopyranosyloxy)-8-fluoro-4-methylcoumarin (1, Neu5Acα2,6GalβFMU) have been synthesized and used to probe the rate-limiting step for turnover by the M. viridifaciens sialidase. The derived kinetic isotope effects (KIEs) on k(cat) for the ring oxygen ((18)V), leaving group oxygen ((18)V), anomeric carbon ((13)V), C3-carbon ((13)V), C3-R deuterium ((D)V(R)), C3-S deuterium ((D)V(S)), and C3-dideuterium ((D)(2)V) are 0.986 ± 0.003, 1.003 ± 0.005, 1.021 ± 0.006, 1.001 ± 0.008, 1.029 ± 0.007, 0.891 ± 0.008, and 0.890 ± 0.006, respectively. The solvent deuterium KIE ((D(2)O)V) for the sialidase-catalyzed hydrolysis of 1 is 1.585 ± 0.004. In addition, a linear proton inventory was measured for the rate of hydrolysis, under saturating condition, as a function of n, the fraction of deuterium in the solvent. These KIEs are compatible with rate-determining cleavage of the enzymatic tyrosinyl β-sialoside intermediate. Moreover, the secondary deuterium KIEs are consistent with the accumulating Michaelis complex in which the sialosyl ring of the carbohydrate substrate is in a (6)S(2) skew boat conformation. These KIE measurements are also consistent with the rate-determining deglycosylation reaction occurring via an exploded transition state in which synchronous charge delocalization is occurring onto the ring oxygen atom. Finally, the proton inventory and the magnitude of the solvent KIE are consistent with deglycosylation involving general acid-catalyzed protonation of the departing tyrosine residue rather than general base-assisted attack of the nucleophilic water.     

Transition state analysis of Vibrio cholerae sialidase-catalyzed hydrolyses of natural substrate analogues

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-β-d-galactopyranosyl-(1→4)-1-thio-β-d-glucopyranoside; Neu5Acα2,3LacβSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacβSPh for the ring oxygen ((18)V/K), leaving group oxygen ((18)V/K), C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6βSPh for C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacβSPh and Neu5Acα2,6LacβSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening ((4)H(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6βSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.     

Kinetics of the thiazolium ion-catalyzed benzoin condensation.

The formation of benzoin (Ph-CHOH-CO-Ph) from two molecules of benzaldehyde, catalyzed by 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide in methanol buffered with Et(3)N/Et(3)NH(+)Cl(-) has been studied. Initial-rate studies at various concentrations of PhCHO (0.1-1.7 M) showed that the reaction is close to being first order in PhCHO. Following the reaction in deuteriomethanol, (1)H NMR spectroscopy allowed rate constants for all three kinetically significant steps to be determined. These show that all three steps are partially rate-determining. A normal deuterium kinetic isotope effect for the overall reaction (k(H)/k(D) approximately 3.4) is observed using PhCDO, and a large inverse solvent isotope effect (k(D)/k(H) approximately 5.9) is observed using deuteriomethanol, consistent with the kinetic scheme presented here.     

Kinetics and mechanism of hydrogen-atom abstraction from rhodium hydrides by alkyl radicals in aqueous solutions

The kinetics of the reaction of benzyl radicals with [L(1)(H(2)O)RhH{D}](2+) (L(1)=1,4,8,11-tetraazacyclotetradecane) were studied directly by laser-flash photolysis. The rate constants for the two isotopologues, k=(9.3±0.6) × 10(7) M(-1) s(-1) (H) and (6.2±0.3) × 10(7) M(-1) s(-1) (D), lead to a kinetic isotope effect k(H)/k(D)=1.5±0.1. The same value was obtained from the relative yields of PhCH(3) and PhCH(2)D in a reaction of benzyl radicals with a mixture of rhodium hydride and deuteride. Similarly, the reaction of methyl radicals with {[L(1)(H(2)O)RhH](2+) + [L(1)(H(2)O)RhD](2+)} produced a mixture of CH(4) and CH(3)D that yielded k(H)/k(D)=1.42±0.07. The observed small normal isotope effects in both reactions are consistent with reduced sensitivity to isotopic substitution in very fast hydrogen-atom abstraction reactions. These data disprove a literature report claiming much slower kinetics and an inverse kinetic isotope effect for the reaction of methyl radicals with hydrides of L(1)Rh.     

Oxidation of alcohol by lipopathic Cr(VI): a mechanistic study

The oxidation kinetics of various aliphatic primary and secondary alcohols having varied hydrocarbon chain length were studied using cetyltrimethylammonium dichromate (CTADC) in dichloromethane (DCM) in the presence of acetic acid and in the presence of a cationic surfactant. The rate of the reaction is highly sensitive to the change in [CTADC], [alcohol], [acid], [surfactant], polarity of the solvents, and reaction temperature. A Michaelis-Menten type kinetics was observed with respect to substrate. The chemical nature of the intermediate and the reaction mechanism were proposed on the basis of (i) observed rate constant dependencies on the reactants, that is, fractional order with respect to alcohol and acid and a negative order with respect to oxidant, (ii) high negative entropy change, (iii) inverse solvent kinetic isotope effect, k(H2O)/k(D2O) = 0.76, (iv) low primary kinetic isotope effect, kH/kD = 2.81, and (v) the k(obs) dependencies on solvent polarity parameters. The observed experimental data suggested the self-aggregation of CTADC giving rise to a reverse micellar system akin to an enzymatic environment, and the proposed mechanism involves the following: (i) formation of a complex between alcohol and the protonated dichromate in a rapid equilibrium, equilibrium constant K = 5.13 (+/-0.07) dm(3) mol(-1), and (ii) rate determining decomposition (k(2) = (7.6 +/- 0.7) x 10(-3) s(-1)) of the ester intermediate to the corresponding carbonyl compound. The effect of [surfactant] on the rate constant and the correlation of solvent parameters with the rate constants support the contribution of hydrophobic environment to the reaction mechanism.     

KINETIC STUDIES ON THE ACID-CATALYZED PUMMERER REACTION OF α-(METHYLSULFINY)ACETOPHENONES

Abstract

The kinetic behavior of the acid-catalyzed Pummerer reaction of α-(methylsulfinyl)acetophenones in dilute hydrochloric acid has been studied in detail. The kinetic data were analyzed in the light of correlations between the reaction rates and acidity functions, activation parameters, solvent isotope effects, polar effects of substituents, etc. Moreover, ¹⁸O-tracer experiments were also carried out using acid media containing H₂ ¹⁸O. Based on these observations, a plausible mechanism for this reaction has been discussed.     

Study on the transesterification of methyl aryl phosphorothioates in methanol promoted by Cd(II), Mn(II), and a synthetic Pd(II) complex

Methanol solutions containing Cd(II), Mn(II), and a palladacycle, (dimethanol bis(N,N-dimethylbenzylamine-2C,N)palladium(II) (3), are shown to promote the methanolytic transesterification of O-methyl O-4-nitrophenyl phosphorothioate (2b) at 25 °C with impressive rate accelerations of 10(6)-10(11) over the background methoxide promoted reaction. A detailed mechanistic investigation of the methanolytic cleavage of 2a-d having various leaving group aryl substitutions, and particularly the 4-nitrophenyl derivative (2b), catalyzed by Pd-complex 3 is presented. Plots of k(obs) versus palladacycle [3] demonstrate strong saturation binding to form 2b:3. Numerical fits of the kinetic data to a universal binding equation provide binding constants, K(b), and first order catalytic rate constants for the methanolysis reaction of the 2b:3 complex (k(cat)) which, when corrected for buffer effects, give corrected (k(cat)(corr)) rate constants. A sigmoidal shaped plot of log(k(cat)(corr)) versus (s)(s)pH (in methanol) for the cleavage of 2b displays a broad (s)(s)pH independent region from 5.6 ≤ (s)(s)pH ≤ 10 with a k(minimum) = (1.45 ± 0.24) × 10(-2) s(-1) and a [lyoxide] dependent wing plateauing above a kinetically determined (s)(s)pK(a) of 12.71 ± 0.17 to give a k(maximum) = 7.1 ± 1.7 s(-1). Br?nsted plots were constructed for reaction of 2a-d at (s)(s)pH 8.7 and 14.1, corresponding to reaction in the midpoints of the low and high (s)(s)pH plateaus. The Br?nsted coefficients (β(LG)) are computed as -0.01 ± 0.03 and -0.86 ± 0.004 at low and high (s)(s)pH, respectively. In the low (s)(s)pH plateau, and under conditions of saturating 3, a solvent deuterium kinetic isotope effect of k(H)/k(D) = 1.17 ± 0.08 is observed; activation parameters (ΔH(Pd)(++) = 14.0 ± 0.6 kcal/mol and ΔS(Pd)(++)= -20 ± 2 cal/mol·K) were obtained for the 3-catalyzed cleavage reaction of 2b. Possible mechanisms are discussed for the reactions catalyzed by 3 at low and high sspH. This catalytic system is shown to promote the methanolytic cleavage of O,O-dimethyl phosphorothioate in CD3OD, producing (CD3O)2P═O(S(-)) with a half time for reaction of 34 min.     

Conformational Study of α‐N‐Acetyl‐D‐Neuraminic Acid by Density Functional Theory

The stable structures of α‐N‐acetyl‐D‐neuraminic acid (Neu5Acα) in the gas phase were studied at the B3LYP level of theory using 6‐31G(d,p) and 6‐31++G(d,p) basis sets. They are classified into five types according to the patterns of the intramolecular hydrogen bond formations. One of the stable structures had intramolecular hydrogen bond network of O₉H_O9 … O₈H_O8 … O?C₁‐O₁H_O1 and O₇H_O7…O?CHN‐C₅ similar to the crystal structure of Neu5Ac‐α‐methyl glycoside methyl ester. The stable structures of Neu5Acα are reasonable for the following sialooligosaccharide ligand studies with respect to the relationship between OH group orientations and intramolecular hydrogen bond formations. The barrier heights for isomerizations between the stable structures were computed to be 2.8 to 6.7 kcal/mol at the B3LYP/6‐31++G(d,p)//B3LYP/6‐31G(d,p) level, which are basic factors for the conformational behavior of Neu5Acα before its interactions with receptors. We also calculated Neu5Acα–4 or 5‐water complexes to take account of the solvent effect on the intramolecular hydrogen bonds in the stable structures. Consequently, the structures of Neu5Acα in the complexes are similar to each other, which is consistent with the known NMR data. Thus, the optimum Neu5Acα‐water complexes are some of the reasonable pseudohydrous Neu5Acα.     

13C and (2)H kinetic isotope effects and the mechanism of Lewis acid-catalyzed ene reactions of formaldehyde

13C and (2)H kinetic isotope effects were determined for the ene reaction of formaldehyde with 2-methyl-2-butene at natural abundance catalyzed by diethylaluminum chloride. The reactive methyl group exhibits a k(12)(C)/k(13)(C) of 1.006-1.009 and a k(H)/k(D) of approximately 1.22-1.23. The latter represents a combination of primary and secondary effects and is consistent with a significant primary deuterium isotope effect. A very close correspondence of the other isotope effects with the equilibrium isotope effects predicted for formation of a model intermediate cation is observed. An intermolecular deuterium isotope effect of 2.0-2.5 was observed under several reaction conditions in the Lewis acid-catalyzed reaction of formaldehyde with d(0)/d(12)-tetramethylethylene. The results are interpreted as supporting the reversible formation of an essentially classical open cation followed by rate-limiting proton transfer.     

Synthesis of α- and β-Methyl NEU5Ac α-(2→8) NEU5Ac Disaccharides

ABSTRACT

Acid hydrolysis of colominic acid, an α-(2→8)-linked oligomer of sialic acid, yielded Neu5Ac α-(2→8) Neu5Ac (di-Neu5Ac) 2 as one of the products. Starting from this disaccharide, it was possible to prepare two potential di-Neu5Ac donors, 5 and 8, as their corresponding 2-chloro derivatives. Subsequent reaction of the donor 8 with methanol as a simple acceptor led to the α- and β-methyl Neu5Ac α-(2→8) Neu5Ac glycosides.     

Mechanisms of acid decomposition of dithiocarbamates. 5. Piperidyl dithiocarbamate and analogues

In this work, the acid cleavage at 25 degrees C in 20% v/v aqueous ethanol of a series of analogues of piperidine dithiocarbamate X(C2H4)2NCS2(-) (X = CH2, CHCH3, NH, NCH3, S, O) was studied. The pH-rate profiles were obtained in the range of H(o)-5 and pH 5. They all presented a dumbell shaped curve with a plateau from which the pH-independent first-order rate constant k(o) (or the specific acid catalysis k(H)) was calculated, in addition to the acid dissociation constant of the free (pKa) and conjugate acid (pK(+)) species of the DTC. LFERs of the kinetically determined pKa and pK(+) versus pKN (pKa of parent amine) were used to characterize the reactive species and the structure of the transition state of the rate-determining step. For X = CH2, CH3CH the values of k(H) agree with those of alkDTCs in the strong base region of the Br?nsted plot of log k(H) versus pKN where the transition state is close to a zwitterion formed by intramolecular water-catalyzed S-to-N proton transfer of the dithiocarbamic acid. However, when X = NH, CH 3N, O, S, the reactive species is the DTC anion, which is as reactive as an arylDTC, and similarly, the pK(+) values correspond to a parent amine that is about 3-4 pK units more basic. The solvent isotope effect indicated that the acid decomposition of these dithiocarbamate anions is specifically catalyzed by a Hydron anchimerically assisted by the heteroatom through a boat conformation.     

Multiple isotope effect study of the acid-catalyzed hydrolysis of methyl formate

Multiple isotope effects have been measured for the acid-catalyzed hydrolysis of methyl formate in 0.5 M HCl at 20 degrees C. The isotope effects in the present investigation include the carbonyl carbon (13k = 1.028 +/- 0.001), the carbonyl oxygen (18k = 0.9945 +/- 0.0009), the nucleophile oxygen (18k = 0.995 +/- 0.001), and the formyl hydrogen ((D)k = 0.81 +/- 0.02). Determination of the carbonyl carbon, carbonyl oxygen, and formyl hydrogen isotope effects was performed via isotopic analysis of residual substrate. However, determination of the oxygen nucleophile isotope effect required analysis of the oxygen atoms of the product (formic acid), which exchange with the solvent (water) under acid conditions. This necessitated measurement of the rate of exchange of these oxygen atoms under the conditions for hydrolysis (k(ex) = 0.0723 min(-1)) and correction of the raw isotope ratios measured during the nucleophile-O isotope effect experiment. These results, along with the previously reported isotope effect for the leaving oxygen (18k = 1.0009) and the ratio of the rate of hydrolysis to that of exchange of the carbonyl oxygen with water (k(h)/k(ex) = 11.3), give a detailed picture of the transition-state structure for the reaction.     

One‐Pot Synthesis of N‐Acetyl‐ and N‐Glycolylneuraminic Acid Capped Trisaccharides and Evaluation of Their Influenza A(H1 N1) Inhibition

Human lung epithelial cells natively offer terminal N‐acetylneuraminic acid (Neu5Ac) α(2→6)‐linked to galactose (Gal) as binding sites for influenza virus hemagglutinin. N‐Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is known to affect hemagglutinin binding in other species. Not normally generated by humans, Neu5Gc may find its way to human cells from dietary sources. To compare their influence in influenza virus infection, six trisaccharides with Neu5Ac or Neu5Gc α(2→6) linked to Gal and with different reducing end sugar units were prepared using one‐pot assembly and divergent transformation. The sugar assembly made use of an N‐phthaloyl‐protected sialyl imidate for chemoselective activation and α‐stereoselective coupling with a thiogalactoside. Assessment of cytopathic effect showed that the Neu5Gc‐capped trisaccharides inhibited the viral infection better than their Neu5Ac counterparts.     

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.     

Ester hydrolysis and enol nitrosation reactions of ethyl cyclohexanone-2-carboxylate inhibited by beta-cyclodextrin

Both the ester hydrolysis and the nitrosation reactions of the enol tautomer of ethyl cyclohexanone-2-carboxylate (ECHC) are investigated in the absence and presence of beta-cyclodextrin (beta-CD). The ester hydrolysis reaction is studied in dilute H2O and D2O solutions of hydrochloric acid and in aqueous buffered solutions of carboxylic acids (acetic acid and its chloro derivatives). The pseudo-first-order rate constant increases with both the [H+] and the total buffer concentration, indicating that the hydrolysis is subject to acid and general base catalysis. Substantial solvent isotope effects in the normal direction (kH/kD > 1) for the acid-catalyzed hydrolysis was observed. Addition of beta-CD strongly slows the hydrolysis reaction. The variation of the observed rate constant (k(o)) with [beta-CD] exhibits saturation behavior, consistent with 1:1 binding between the enol of ECHC and beta-CD. The binding is quite strong, and bound ECHC-enol is unreactive. The nitrosation reaction of ECHC in aqueous acid medium, using sodium nitrite in great excess over the concentration of ECHC, yields perfect first-order kinetics, indicating that the slow step is the nitrosation of the enol tautomer. This finding suggests that a great percentage of the total ECHC concentration must exist in the enol form. The nitrosation reaction is of first order in [nitrite] and is catalyzed by the presence of Cl-, Br-, or SCN- ions, which indicates that the attack of the nitrosating agent is the slow step. The nitrosation reaction is also strongly inhibited by the presence of beta-CD because of the formation of unreactive inclusion complexes between the host, beta-CD, and the guest, the enol of ECHC. In alkaline medium, the formation of the enolate ion is observed, which absorbs at higher wavelengths (lambda(max) = 256 nm in acid medium shifts to lambda(max) = 288 nm in alkaline medium). This anion also undergoes ester hydrolysis spontaneously, but shows neither specific basic catalysis nor appreciable effect by the presence of beta-CD. From kinetic and spectroscopic measurements the pKa of the enol of ECHC has been determined as 12.35.     

Kinetics and mechanism of hydration of o-thioquinone methide in aqueous solution. Rate-determining protonation of sulfur

o-Thioquinone methide, 2, was generated in aqueous solution by flash photolysis of benzothiete, 1, and rates of hydration of this quinone methide to o-mercaptobenzyl alcohol, 3, were measured in perchloric acid solutions, using H2O and D2O as the solvent, and also in acetic acid and tris(hydroxymethyl)methylammonium ion buffers, using H2O as the solvent. The rate profiles constructed from these data show hydronium-ion-catalyzed and uncatalyzed hydration reaction regions, just like the rate profiles based on literature data for hydration of the oxygen analogue, o-quinone methide, of the presently examined substrate. Solvent isotope effects on hydronium-ion catalysis of hydration for the two substrates, however, are quite different: k(H)/k(D) = 0.42 for the oxygen quinone methide, whereas k(H)/k(D) = 1.66 for the sulfur substrate. The inverse nature (k(H)/k(D) < 1) of the isotope effect in the oxygen system indicates that this reaction occurs by a preequilibrium proton-transfer reaction mechanism, with protonation of the substrate on its oxygen atom being fast and reversible and capture of the benzyl-type carbocationic intermediate so formed being rate-determining. The normal direction (k(H)/k(D) > 1) of the isotope effect in the sulfur system, on the other hand, suggests that protonation of the substrate on its sulfur atom is in this case rate-determining, with carbocation capture a fast following step. A semiquantitative argument supporting this hypothesis is presented.     

3 beta-Hydroxysialic acid glycosides. I. Calcium-binding ability and chemical and enzymatic stabilities.

Methyl alpha- and beta-glycosides of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-3 beta-hydroxyneuraminic acid (Neu5Ac beta 3OH) (1-4) were prepared to evaluate their calcium-binding ability. (Methyl alpha-glucopyranosidonyl) alpha- and beta-, and 4-methylumbelliferyl alpha-glycosides of Neu5Ac and Neu5Ac beta 3OH (5-10) were also synthesized for the comparison of chemical and enzymatic stabilities, respectively. Methyl beta-glycosides of Neu5Ac and Neu5Ac beta 3OH, 3 and 4, respectively, showed intense calcium-binding abilities, while no such ability was observed in the corresponding alpha-glycosides, 1 and 2. The Neu5Ac beta 3OH glycosides, 6, 8, and 10, showed much stronger resistance to acidic hydrolysis and sialidase digestion than the corresponding Neu5Ac glycosides, 5, 7, and 9.     

Reactivity of phthalimide N-oxyl radical (PINO) toward the phenolic O-H bond. A kinetic study

The reactivity of the phthalimide N-oxyl radical (PINO) toward the OH bond of a series of substituted phenols was kinetically investigated in CH(3)CN. The reaction selectivity and the deuterium kinetic isotope effect were determined. Information on the kinetic solvent effect was also obtained with phenol as the substrate.     

Decay of the peroxide intermediate in laccase: reductive cleavage of the O-O bond.

Laccase is a multicopper oxidase that contains four Cu ions, one type 1, one type 2, and a coupled binuclear type 3 Cu pair. The type 2 and type 3 centers form a trinuclear Cu cluster that is the active site for O(2) reduction to H(2)O. To examine the reaction between the type 2/type 3 trinuclear cluster and dioxygen, the type 1 Cu was removed and replaced with Hg(2+), producing the T1Hg derivative. When reduced T1Hg laccase is reacted with dioxygen, a peroxide intermediate (P) is formed. The present study examines the kinetics and mechanism of formation and decay of P in T1HgLc. The formation of P was found to be independent of pH and did not involve a kinetic solvent isotope effect, indicating that no proton is involved in the rate-determining step of formation of P. Alternatively, pH and isotope studies on the decay of P revealed that a proton enhances the rate of decay by 10-fold at low pH. This process shows an inverse k(H)/k(D) kinetic solvent isotope effect and involves protonation of a nearby residue that assists in catalysis, rather than direct protonation of the peroxide. Decay of P also involves a significant oxygen isotope effect (k(16)O(2)/k(18)O(2)) of 1.11 +/- 0.05, indicating that reductive cleavage of the O-O bond is the rate-determining step in the decay of P. The activation energy for this process was found to be approximately 9.0 kcal/mol. The exceptionally slow rate of decay of P is explained by the fact that this process involves a 1e(-) reductive cleavage of the O-O bond and there is a large Franck-Condon barrier associated with this process. Alternatively, the 2e(-) reductive cleavage of the O-O bond has a much larger driving force which minimizes this barrier and accelerates the rate of this reaction by approximately 10(7) in the native enzyme. This large difference in rate for the 2e(-) versus 1e(-) process supports a molecular mechanism for multicopper oxidases in which O(2) is reduced to H(2)O in two 2e(-) steps.     

Base-Catalyzed Dehydration of 3-Substituted Benzene cis-1,2-Dihydrodiols: Stabilization of a Cyclohexadienide Anion Intermediate by Negative Aromatic Hyperconjugation

Evidence that a 1,2-dihydroxycyclohexadienide anion is stabilized by aromatic "negative hyperconjugation" is described. It complements an earlier inference of "positive" hyperconjugative aromaticity for the cyclohexadienyl cation. The anion is a reactive intermediate in the dehydration of benzene cis-1,2-dihydrodiol to phenol. Rate constants for 3-substituted benzene cis-dihydrodiols are correlated by σ(-) values with ρ = 3.2. Solvent isotope effects for the reactions are k(H(2)O)/k(D(2)O) = 1.2-1.8. These measurements are consistent with reaction via a carbanion intermediate or a concerted reaction with a "carbanion-like" transition state. These and other experimental results confirm that the reaction proceeds by a stepwise mechanism, with a change in rate-determining step from proton transfer to the loss of hydroxide ion from the intermediate. Hydrogen isotope exchange accompanying dehydration of the parent benzene cis-1,2-dihydrodiol was not found, and thus, the proton transfer step is subject to internal return. A rate constant of ～10(11) s(-1), corresponding to rotational relaxation of the aqueous solvent, is assigned to loss of hydroxide ion from the intermediate. The rate constant for internal return therefore falls in the range 10(11)-10(12) s(-1). From these limiting values and the measured rate constant for hydroxide-catalyzed dehydration, a pK(a) of 30.8 ± 0.5 was determined for formation of the anion. Although loss of hydroxide ion is hugely exothermic, a concerted reaction is not enforced by the instability of the intermediate. Stabilization by negative hyperconjugation is proposed for 1,2-dihydroxycyclohexadienide and similar anions, and this proposal is supported by additional experimental evidence and by computational results, including evidence for a diatropic ("aromatic") ring current in 3,3-difluorocyclohexadienyl anion.